The polypeptide hirudin, originally isolated from the leech Hirudo medicinalis, is a highly specific thrombin inhibitor of broad therapeutic potential. F. Markwardt, Biomed. Biochim. Acta 44: 1007-1013 (1985). However, the amounts required can only be produced by genetic engineering methods by means of transformed microorganisms. It has been found that the yeast Saccharomyces cerevisiae is suitable as a host organism for producing correctly folded and fully active hirudin. EP A1 168 342; EP A1 200 655.
The yeast Saccharomyces cerevisiae, which is particularly preferably used for producing recombinant peptides and proteins by genetic engineering, forms the enzyme carboxypeptidase Y (CPY). CPY is a thermally unstable molecule. See Kuhn et al., "Isolation and Partial Characterisation of an Acid Carboxypeptidase from Yeast," Biochemistry, 13(19): 3871-77 (1974). A 5-minute heat treatment at 68.degree. C. and pH=7.0 leads to complete inactivation of CPY, and lower temperatures lead to partial inactivation of CPY.
This enzyme non-specifically cleaves off various amino acids from the C terminus of proteins and can therefore be used for determining the carboxy-terminal sequence of proteins. Obviously, this enzyme is also able to break down proteins of value expressed in the yeast, including pharmacologically active compounds such as hirudin, during the production process, in particular during chromatographic purification. This leads to the formation of by-products and losses of yield, which can be significant. In addition, CPY can reduce the stability of high-purity products if it is still present in traces in a preparation, e.g., if it is present in a lyophilizate.
Hirudin has a free C terminus and is therefore exposed substantially unprotected to enzymatic attack by CPY.
One solution to this problem is to use mutant yeast strains in which the CPY activity is suppressed or eliminated by various methods. See, for example, EP 390 676, EP 341 215 and GB 2 249 096. However, these methods necessitate a relatively great change in the yeast strain which may possibly restrict the growth behavior and/or alter the physiological stability of the microorganisms.
CPY can also be inhibited by chemical inhibitors, e.g., by phenylmethylsulfonyl fluoride (PMSF). A chemical inhibition of this type is undesirable, at least in the case of pharmaceutically active compounds, since it can lead to partial derivatization which is sometimes undetectable.
Thus, there is a need for a process for inactivating CPY in hirudin-containing culture broths that does not have the drawbacks of the above-mentioned methods. There also is a need for a means for performing this process on a large scale, for example, on an industrial scale.